Institute of Mechanobiology and Medical Engineering,Shanghai Jiao Tong University
目的 探究周期性高张应变和血小板源性微囊(platelet-derived microvesicles,PMVs)对血管平滑肌细胞(vascular smooth muscle cells,VSMCs)迁移功能的影响,以及钙离子在其中的相关机制。方法 应用FX-5000T细胞应变加载系统对体外培养的VSMCs施加5%和15%幅度周期性张应变,分别模拟VSMCs受到的生理条件的周期性张应变和高血压条件下的周期性高张应变；使用划痕实验检测VSMCs迁移；使用无钙离子培养基创造细胞外无游离钙离子条件；应用IP3R拮抗剂2-APB阻断细胞内钙离子释放；施加TRPV4通道拮抗剂GSK219和L型电压门控钙通道的抑制剂Nifedipine分别阻断相应钙离子通道；体外施加凝血酶激活血小板活化产生PMVs以模拟高血压病理环境。结果 与5%周期性张应变相比,15%周期性张应变可以显著促进VSMCs迁移。去除细胞外钙离子可以抑制VSMCs迁移,但GSK219和Nifedipine对于15%高张应变诱导的VSMCs迁移无显著作用；2-APB可以抑制15%高张应变诱导的VSMCs迁移过程。同时PMVs可以显著促进15%高张应变诱导的VSMCs迁移,并且细胞外和细胞内钙离子均参与其中。结论 细胞内钙和细胞外钙在15%周期性高张应变诱导的VSMCs迁移中均发挥重要作用,并且PMVs可以协同参与到上述过程中。本研究旨在为高血压病理性张应变诱导血管重建的分子机制和临床治疗提供力学生物学新思路。
Objective To investigate the synergistic effects of pathologically elevated cyclic stretch and platelet-derived microvesicles (PMVs) on migration of vascular smooth muscle cells (VSMCs) and the potential role of calcium in this process. Methods The FX-5000T cell strain loading system was applied to VSMCs with magnitude of 5% and 15% cyclic stretch to simulate physiological and hypertensive situation respectively in vitro; Wound healing assay was used to analyze the migration of VSMCs; Ca2+-free medium was used to remove extracellular calcium; 2-APB (an antagonist of IP3R) was used to inhibit the release of intercellular stored calcium; GSK219 (an antagonist of TRPV4), and Nifedipine (an inhibitor of L-type voltage-gated calcium channel) were applied to block the activity of respective calcium channel; Thrombin was used to stimulate platelets in vitro which mimics the hypertensive activation of PMVs in vivo. Results Compared with 5% cyclic stretch, 15% cyclic stretch significantly promoted VSMC migration. Removal of extracellular calcium inhibited the migration of VSMCs, but the application of GSK219 and Nifedipine did not affect the migration upregulated by 15% cyclic stretch; while 2-APB which inhibited the release of intracellular stored calcium could also repress the migration of VSMCs during 15% cyclic stretch application. PMVs further promoted VSMC migration under 15% cyclic stretch condition, and both extracellular calcium and intercellular stored calcium were involved in this process. Conclusion The research revealed that both intracellular and extracellular calcium play important roles in VSMC migration induced by 15% cyclic stretch, and PMVs synergistically participate in the above process. The study is aim to provide new mechanobiological insights into the molecular mechanism and clinical targets of vascular remodeling in hypertension.